The heterodisulfide reductase (HDR) from Methanosarcina thermophila was purified to homogeneity from acetate-grown cells. In the absence of detergents, HDR consisted of an eight-protein complex with hydrogenase activity. However, when HDR was purified in the presence of 0.6% Triton X-100, a two-subunit (53 and 27 kDa) high specific activity ( approximately 200 units mg-1) enzyme was obtained that lacked hydrogenase activity. Sedimentation equilibrium experiments demonstrated that HDR has a molecular mass of 206 kDa and a high partial specific volume (0.9 cm3/g), indicating that the purified protein contains a significant amount of bound lipid. Like the HDR from Methanosarcina barkeri [Kunkel, A., Vaupel, M., Heim, S., Thauer, R. K., and Hedderich, R. (1997) Eur. J. Biochem. 244, 226-234], it was found to contain two discrete b-type hemes in the small subunit and two distinct [Fe4S4]2+/1+ clusters in the large subunit. One heme is high-spin and has a high midpoint potential (-23 mV), whereas the other heme apparently is low-spin and exhibits a relatively low midpoint potential (-180 mV). Only the high-spin heme binds CO. The midpoint potentials for the two clusters are -100 and -400 mV. In the fully reduced state, a complicated EPR spectrum with g values of 2.03, 1.97, 1.92, and 1.88 was observed. This spectrum resembles that of 8Fe ferredoxins in the fully reduced state, indicating that the two clusters in HDR are near enough to experience relatively strong dipolar interactions. Kinetic studies in which CO oxidation is coupled to heterodisulfide reduction strongly indicate that a membrane-associated compound is the direct electron donor to HDR. An electron-transfer pathway is presented that postulates a mechanism for coupling electron transport to proton translocation.